The present invention relates to a human ADAMTS-1 protein, a gene encoding the same, a pharmaceutical composition, and a method for immunologically analyzing the human ADAMTS-1 protein.
A mouse ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs)-1 gene has been cloned as a cDNA from a mouse colon cancer cell which induces cancer cachexia when transplanted to a mouse. The mouse ADAMTS-1 protein encoded by the gene is a unique protein containing a matrix metalloproteinase domain, a disintegrin domain, and three thrombospondin domains [J. Biol. Chem., 272, 556-562 (1997)]. The physiological functions of the mouse ADAMTS-1 protein has not been reported, but there have been reports of each individual functional domain contained therein.
For example, a snake venom disintegrin belongs to a family of proteins which are rich in cystein, and exhibit an anticoagulant activity [Semin. Hematol., 31, 289-300 (1994)].
Further, for example, an ADAM (A disintegrin and metalloproteinase) family is known as a protein family containing a matrix metalloproteinase domain and a disintegrin domain [Nature, 377, 652-656 (1995); Nature Genet., 5, 151-157 (1993); Nature, 356, 248-252 (1992)]. Examples of known ADAM family proteins are fertilin, epidermal apical protein, cyritestin, MDC (metalloprotease-like, disintegrin-like and cystein-rich protein), meltrin, MS2, and metargidin [Nature, 377, 652-656 (1995); Nature Genet. 5, 151-157 (1993); Nature, 356, 248-252 (1992); Biochem. J., 286, 671-675 (1992); Dev. Growth. Differ., 36, 49-58 (1994); Int. Immunol., 2, 585-591 (1990); J. Biol. Chem., 271, 4593-4596 (1996)].
It was reported that fertilin is involved in an integrin-mediated sperm-egg binding [Nature, 356, 248-252 (1992)], and meltrin is involved in a myotube formation [Nature, 377, 652-656 (1995)]. MDC expressed mainly in a central nervous system is a candidate as a suppresser against a human breast cancer [Nature Genet. 5, 151-157 (1993)], and MS2 serves as a macrophage antigen [Int. Immunol., 2, 585-591 (1990)]. However, little have been known about the physiological roles of these ADAM family proteins.
The mouse ADAMTS-1 protein contains a matrix metalloproteinase domain and a disintegrin domain, and therefore belongs to the ADAM family. However, the mouse ADAMTS-1 protein is different from other known ADAM family proteins in that it further contains thrombospondin domains.
As mentioned, the ADAM family proteins have various kinds of activities such as the involvement of bone or muscle metabolism, suppression of cancer growth, or fertilization, and the thrombospondin exhibits an action to inhibit vascularization and suppresses cancer. Therefore, it is expected that the mouse ADAMTS-1 protein will exhibit unique physiological functions.
The inventors of the present invention attempted to isolate the corresponding human ADAMTS-1 protein. Accordingly, the present inventors designed and prepared various probes on the basis of the base sequence of the known mouse ADAMTS-1 gene, and carried out plaque hybridizations with a human kidney cDNA library so as to obtain a human ADAMTS-1 gene, but a desired gene was not obtained. Then, the present inventors designed and prepared various primers on the basis of the base sequence of the known mouse ADAMTS-1 gene, and carried out PCRs, using the human kidney cDNA library as templates under ordinary conditions, so as to obtain the desired gene, but did not succeed.
Thereafter, the present inventors carried out a PCR of the human kidney cDNA library, using the same primers, but under conditions milder than those ordinarily used, more particularly, an annealing temperature is set lower than an ordinary temperature, and the inventors successfully obtained a novel human ADAMTS-1 gene. The resulting gene was then, expressed in E. coli, and the biological activities of the recombinant human ADAMTS-1 protein were examined. Surprisingly, it was found that the novel human ADAMTS-1 protein can decrease the numbers of leukocytes and platelets, and at the same time, increase the number of erythrocytes. Such activities in influencing hematopoietic functions cannot be expected from the structure of the mouse ADAMTS-1 gene which was used as the basis in designing the primers, or from the functions of domains contained in the human ADAMTS-1 protein. The present invention is based on the above findings.
The present invention relates to a protein characterized by containing an amino acid sequence of SEQ ID NO: 1:
Further, the present invention relates to the variation of proteins which are equivalent to the protein containing the amino acid sequence of SEQ ID NO: 1.
Further, the present invention relates to a protein characterized by containing a matrix metalloproteinase domain, a disintegrin domain, and a thrombospondin domain, except for a mouse ADAMTS-1 protein.
Further, the present invention relates to a gene characterized by encoding the above novel proteins.
Further, the present invention relates to a vector characterized by containing the above gene.
Further, the present invention relates to a transformant characterized by being transformed by the above vector.
Further, the present invention relates to a pharmaceutical composition characterized by comprising (1) the protein containing the amino acid sequence of SEQ ID NO: 1, (2) the variation functionally equivalent to the protein containing the amino acid sequence of SEQ ID NO: 1, or (3) the protein containing a matrix metalloproteinase domain, a disintegrin domain, and a thrombospondin domain.
Further, the present invention relates to an immunologically reactive substance (such as a polyclonal antibody or a monoclonal antibody, or an antibody fragment thereof, or an antiserum) characterized by nature of being capable of specifically reacting with the novel proteins.
Further, the present invention relates to a method for immunologically analyzing the human ADAMTS-1 protein, characterized in that a sample is brought into contact with the immunologically reactive substance, and a complex of the human ADAMTS-1 protein and the immunologically reactive substance is detected.
Further, the present invention relates to a method for analyzing an mRNA of the human ADAMTS-1 protein, characterized in that a sample is brought into contact with a polynucleotide containing a base sequence complementary to that of the mRNA of the human ADAMTS-1 protein consisting of the amino acid sequence of SEQ ID NO: 1, and a complex of the mRNA of the human ADAMTS-1 protein and the polynucleotide is detected.
Further, the present invention relates to a method for extracorporeally detecting an immunological state, characterized by analyzing the human ADAMTS-1 protein or the mRNA thereof.
Further, the present invention relates to an agent for analyzing an immunological state, characterized by containing the immunologically reactive substance capable of immunologically reacting the human ADAMTS-1 protein or the polynucleotide containing the base sequence complementary to that of the mRNA of the human ADAMTS-1 protein.